In the life cycle of HIV, the Rev protein regulates the temporal switch from the early regulatory to the late lytic phase by binding to a highly structured RRE (Rev Responsive Element) RNA. Previously, we reported on the kinetic and stoichiometric parameters of this interaction in vitro using a Surface Plasmon Resonance (SPR) biosensor. Now, we have defined the minimal structural requirements for a functionally competent Rev using mutants that deleted portions of the N-terminal region of Rev in the context a Rev/MS-C fusion protein that could be targeted to RRE or MS2 RNA. Rev truncated to between residues 19 and 87 still retained most of the Rev function. We showed that under conditions of stringent specificity, a minimal Rev peptide peptide encompassing residues 22-85 and containing the effector motif retained fidelity of Rev function in vivo albeit with reduced in vitro binding affinity for RRE RNAs. We further show that a Rev mutant that deleted residues 18-24 in the context of the Rev/MS-C fusion protein had a null phenotype and was a novel trans-dominant inhibitor of Rev. We analyzed the role(s) of the arginine rich RNA binding domain in RNA binding and trans-activation. The arginine rich RNA binding domain of Rev in the fusion protein could be functionally replaced by a stretch of 9 arginines. However, poly-arginine substitutions expanded the RNA binding specificity of the resultant mutant Rev protein. Among the accessory proteins encoded by HIV-1, the 27 kDal membrane associated myristoylated Nef has proved to be enigmatic. However, there is reasonable agreement in the literature on the potential roles of Nef in modulating certain cellular receptors. Nef induced a 8-30 fold and a 4-20 fold down-modulation of cell surface CD4 and CCR5 respectively. The functional consequence of this effect was confirmed by ligand CCR5 ligand (RANTES or MIP-1beta) induced receptor phosphorylation and calcium flux. However, even with the maximal levels of Nef effect, there was no significant effect on the virus entry of M-tropic strains of HIV-1. Nef effect was dose dependent, and certain HIV nef alleles and SIV Nef were quite potent in this respect. CCR5 mutants with truncated tails were downregulated by Nef, if they are expressed on the cell surface. CCR5 tail mutants that were devoid of phosphorylation and signaling phenotypes were still sensitive to Nef effect. In contrast to the effect on CCR5, SIV Nef induced downregulation of CXCR4 resulted in corresponding suppression of virus entry by the T-tropic strains of HIV-1. These results led us to conclude that the threshold of CCR5 for HIV entry is lower than that of CXCR4 and propose that Nef may act as a tropism switching factor during natural infection.We examined the cell surface expression, chemokine dependent receptor phosphorylation, and down-stream signaling events (measured by Ca++ flux rates) and usage by macrophage tropic HIV of wt human CCR5 and mutant derivatives. Tail-less mutants of CCR5 or nested C-terminal deletions for residues up to the 4th trans-membrane domain, and mutants substituting all the cationic residues in the tail for alanines failed to be expressed at the cell surface. Fine-structure mapping of the cytoplasmic tail showed that as few as four residues were sufficient to restore the cell surface expression. CCR5 tail deletions that were competent for cell surface expression and the Ser/Thr substitution mutant also supported macrophage tropic HIV entry. We also evaluated the potential of transport defective CCR5 mutants for inhibiting the functional expression of wt CCR5 by subunit mixing and defective hetero-oligomer formation. When molar excess of defective CCR5s were co-expressed with the wt receptor, subunit mixing was demonstrable by co-immunopreciptation of wt and mutant CCR5s. However, over-expression of defective CCR5s (including the well characterized D32 mutant) did not materially alter the surface presentation, chemokine mediated receptor phosphorylation or Ca++ mobilization, or usage by macrophage tropic HIV of the co-expressed wt CCR5.